Abstract

Currently, the field of mammalian membrane protein structural biology is in its infancy. Existing technologies and experiences have shown that it is possible to obtain the structures of mammalian membrane proteins if sufficient work and thought has been invested. However, there is still an urgent need to develop new methodologies and approaches to improve all aspects of this important area of biological research. Here, a series of novel technologies for the overproduction, purification and crystallisation of human membrane proteins are described which have been tested with a representative member from each of the G-protein coupled receptor (adenosine 2a receptor (A2aR)) and membrane enzyme (sterol isomerase (SI)) superfamilies.
The methylotrophic yeast Pichia pastoris is an excellent host cell for the overproduction of recombinant proteins including membrane proteins of mammalian origin. However, the commercially available expression vectors are far from what is required to maximise the production levels as well as simplify
the detergent extraction and purification of human membrane proteins. Here, a series of related expression constructs were made that had different combinations of tags at both ends of the recombinant protein. The final optimised expression vectors had a C3 protease-iLOV-biotin acceptor-His10 (CLBH) tag fused to the C-terminus of the recombinant protein. The -CLBH vectors gave high level production of both test proteins (one Nin – hSI; one Nout – hA2aR) that could be rapidly purified to homogeneity using a generic protocol. The position of the His10 tag did not affect the expression level of the recombinant protein. In contrast, fusion of the biotin acceptor domain to the C-terminus of the recombinant protein increased its expression by a factor of between 2-4. The
biotin acceptor domain could also be fully biotinylated in vitro using recombinantly expressed biotin ligase allowing purification/immobilisation of the target protein with streptavidin beads. Removal of the expression/ purification tags from the recombinant proteins with C3 protease occurred more efficiently
than when TEV protease was used. An optimised protocol was developed that gave maximal production of our target proteins in fermenter culture at an induction temperature of 22°C. Care was
taken to find a methanol feed rate that gave the highest levels of protein production without causing the accumulation of excess methanol in the culture (which is known to be toxic to the yeast). Using this protocol it was possible to make both hSI and hA2aR with a production level >10 mg of recombinant protein
per litre of culture. As most MPs are colourless, target protein identification is usually performed by methods such as radioligand binding and/or Western blotting. However, these techniques can be time-consuming, use a lot of protein and do not give any
information on the aggregation state of the protein in detergent solution. Previously, it has been shown that the processes of identifying and analysing membrane proteins in detergent solution can be accelerated by attaching green fluorescent
protein to the C-terminus of the recombinant MP. Here, the potential of the recently described iLOV fluorescence tag for membrane protein applications was assessed. iLOV was shown to be an useful tool for optimising processes such as yeast clonal selection, protein production in fermenter culture, detergent and construct screening as well as tracking recombinant MPs through
the purification process. Of note, the iLOV tag allowed a direct assessment of the stability and dispersity state of both target MPs in a range of detergents by fluorescence size exclusion chromatography (FSEC). Using this approach, it was
shown that wild-type hA2aR solubilised using a combination of dodecyl-βDmaltoside (DDM) and cholesteryl-hemisuccinate (CHS) aggregated during purification on a Ni2+ column. Furthermore, it was shown that the hA2aR agonistconformationally-fixed mutant Rag23 is stable in DDM without any CHS present.
Moreover, Rag23 was found to be monodisperse in a series of short-chain detergents (decyl-βD-maltoside, nonyl-βD-maltoside (NM) and β-octylglucoside) suggesting that this mutant is well-suited to structural studies. SI was remarkably robust in short chain detergents demonstrating a reasonable level of stability in
the short chain detergent NM. The FSEC experiments showed that wild-type SI has considerably higher intrinsic stability than native hA2aR suggesting that membrane enzymes will prove to be more amenable to structural analysis than GPCRs.
Rag23 and SI were both purified to homogeneity in a simple four-step procedure: i) Ni2+ purification, ii) cleavage with C3 protease, iii) reverse Ni2+ purification and iv) gel-filtration chromatography. A buffer/salt screen was devised that allowedthose conditions where SI had maximal thermostability in detergent-solution to be
identified. SI was found to have greatest stability in sodium phosphate buffer at acidic pH. Using this information, it was possible to purify monodisperse SI in DM suggesting that this protein may make an excellent candidate for structural
studies too. Crystallisation trials with SI were performed using the commercially available sparse matrix screen MemSys/MemStart. In addition, a lipidic-sponge phase sparse-matrix crystallisation screen that was developed in collaboration with Prof. Richard Neutze (University of Chalmers, Sweden) was tested using SI.
Cholesterol could be incorporated into all of the sponges that make up the screen upto a concentration of 10%. (This is important as the activity of many mammalian membrane proteins is cholesterol-dependent). To date, no diffracting crystals of SI have been obtained with either the conventional or lipidic-sponge
phase crystallisation approaches. In short, a series of novel technologies/methodologies have been developed that will act as a platform for future efforts to solve the structures of a wide-range of human membrane proteins.